Emmy Abbey

Topic

Fluorescence Excitation Emission Matrix Spectral Imaging (FLEEMSI) Microscopy

Department of Chemistry

Date & location

• Thursday, May 14, 2026

• 10:00 A.M.

• Elliott Building

• Room 226

Reviewers

Supervisory Committee

• Dr. Peter Loock, Department of Chemistry, University of Victoria (Supervisor)

• Dr. Alexandre Brolo, Department of Chemistry, UVic (Member)

• Dr. Julian Smazynski, Department of Biochemistry and Microbiology, UVic (Outside Member) 

External Examiner

• Dr. Russ Algar, Department of Chemistry, University of British Columbia 

Chair of Oral Examination

• Dr. John Burke, Department of Biochemistry and Microbiology, UVic 

Abstract

With this dissertation the capabilities of fluorescence imaging—specifically fluorescence microscopy—are greatly extended to allow for chemical fingerprinting in each pixel of an image. Spatial fluorescence imaging microscopy using multispectral and hyperspectral cameras with multiplexed excitation sources has been thus far not described in the literature. Three systems to acquire fluorescence excitation-emission matrix (F-EEM) spectra are demonstrated in this work, using Hadamard-multiplexed programmable excitation light sources. Computational approaches to bypass the limitations of multi- and hyperspectral camera hardware are implemented to increase the time resolution and emission spectral resolution.

The first chapter contains background and instrumentation used in F-EEM spectroscopy and multispectral imaging. Microscopy illumination methods are described as they relate to the methods used in this work, and a brief overview of parameters used for creating spatially distinct dye samples under a microscope are described. Raman spectroscopy and how it relates to multiplexed fluorescence spectroscopy is described and is followed by an overview of multivariate analysis methods used in F-EEM analysis.

F-EEM imaging requires a fully programmable excitation light source and an imaging detector. The work shown here develops and uses Hadamard-multiplexed excitation sources based on instruments built by our group in the past. The basis for multiplexed spectroscopy and the theory of the Hadamard transform are described in Chapter 2. The optical design and software integration of two programmable light sources—one white-light source based on a digital micromirror array and one using an array of discrete-wavelength laser diodes—are described in Chapter 3.

Acquisition of F-EEM images is done through one of two cameras—a snapshot multispectral camera using an eight-channel colour filter array, or an interferogram-based hyperspectral camera providing up to 141 spectral channels over a wide wavelength range. These commercial cameras and their integration into our systems are described in Chapter 4.

Chapter 5 describes the analysis of F-EEM images acquired using a Hadamard-modulated light source and the steps which are unique to imaging and large datasets.

The use of a programmable white-light source for fluorescent component separation is demonstrated in Chapter 6, where an image of capillaries containing fluorophores and mixtures thereof is analyzed using multivariate analysis to demonstrate the spatial and spectral separation of four fluorescent components in an image.

The excitation light source is then modified for use in numerous microscopy illumination methods and F-EEM microscopic imaging using three distinct combinations of excitation source and imaging detectors, is demonstrated in Chapter 7 using combinations of dye emulsions. In Chapter 8, spectral upscaling of an eight-channel emission spectrum acquired with the multispectral camera is demonstrated to be a superior method of fluorophore identification and separation in an F-EEM image. We also apply a new computational method for increasing the time resolution of F-EEM images acquired using a Hadamard-modulated excitation light source. The combination of these two computational techniques allows us to obtain chemical identifiers and concentrations for each of the 65,536 pixels per frame, when these frames are obtained at a rate between 3-10 per second.

Future work for a multiplexed Raman spectroscopy experiment and numerous avenues for experiments using the programmable light sources with the multispectral and hyperspectral cameras are described in Chapter 9.